Methods and Compositions for Identifying Yeast

ABSTRACT

The invention relates to a method of identifying a specific yeast species in patient tissue or body fluid. The method comprises the steps of extracting and recovering DNA of the yeast species from the patient tissue or body fluid, amplifying the DNA, hybridizing a probe to the DNA to specifically identify the yeast species, and specifically identifying the yeast species. The invention also relates to a method of identifying a yeast mycotoxin in patient tissue or body fluid. The method comprises the steps of extracting and recovering the yeast mycotoxin from the patient tissue or body fluid, contacting the yeast mycotoxin with an antibody directed against the yeast mycotoxin, and identifying the yeast myocotoxin. Both of these methods can be used to determine if a patient is at risk for or has developed a disease state related to a yeast infection, and to develop an effective treatment regimen for the patient.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. §119(e) to U.S.Provisional Application Ser. No. 61/091,188, filed on Aug. 22, 2008,incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

This invention relates to methods and compositions for detecting oridentifying yeast and mycotoxins. More particularly, the inventionrelates to methods and compositions for detecting or identifying yeastand mycotoxins in the tissues or body fluid samples of patients.

BACKGROUND AND SUMMARY

Infections by various yeast species have become recognized as a cause ofdisease and ultimate death in many types of patients, including cancerpatients and immunocompromised patients. Candidemia has increased overthe past decade with Candida albicans being the most often isolatedorganism. Other species frequently isolated from infected patientsinclude Candida glabrata, Candida tropicalis, and Candida krusei.Resistance to therapeutics, including the polyenes and the azoles, iscommon in patients infected with various yeast species.

A sensitive method using PCR assays which are based on the detection ofyeast DNA in human body fluids or tissue samples is herein described.The use of realtime PCR (RT-PCR) for detection of yeast infectionsincreases the reliability of PCR results.

In one embodiment, a method of identifying a specific yeast species inpatient tissue or body fluid is provided. The method comprises the stepsof extracting and recovering DNA of the yeast species from the patienttissue or body fluid, amplifying the DNA, hybridizing a probe to the DNAto specifically identify the yeast species, and specifically identifyingthe yeast species.

In this embodiment, 1) the amplifying step can be performed with primersthat hybridize to the DNA, 2) the body fluids can be selected from thegroup consisting of urine, nasal secretions, nasal washes, bronchiallavages, bronchial washes, spinal fluid, sputum, gastric secretions,seminal fluid, other reproductive tract secretions, lymph fluid, wholeblood, serum, and plasma, 3) the DNA can be amplified using PCR, 4) thePCR can be real-time PCR, 5) the probe can be fluorescently labeled, 6)the yeast species can be selected from the group consisting of Candidaalbicans, Candida glabrata, Candida kruseii, and Candida tropicalis, 7)the probe, a forward primer, and a reverse primer can be used during theamplification step and the probe comprises the sequence of SEQ ID NO: 1,the forward primer comprises the sequence of SEQ ID NO: 2, and thereverse primer comprises the sequence of SEQ ID NO: 3, 8) the probe, aforward primer, and a reverse primer can be used during theamplification step and the probe comprises the sequence of SEQ ID NO: 4,the forward primer comprises the sequence of SEQ ID NO: 5, and thereverse primer comprises the sequence of SEQ ID NO: 6, 9) the probe, aforward primer, and a reverse primer can be used during theamplification step and the probe comprises the sequence of SEQ ID NO: 7,the forward primer comprises the sequence of SEQ ID NO: 8, and thereverse primer comprises the sequence of SEQ ID NO: 9, 10) the probe, aforward primer, and a reverse primer can be used during theamplification step and the probe comprises the sequence of SEQ ID NO:10, the forward primer comprises the sequence of SEQ ID NO: 11, and thereverse primer comprises the sequence of SEQ ID NO: 12, 11) theamplified sequence can be internal transcribed spacer regions of nuclearribosomal DNA, and/or 12) the probe can be bound to a bead dyed with afluorochrome. Any applicable combination of 1 through 12 is alsocontemplated.

In another embodiment, a method of identifying a yeast mycotoxin inpatient tissue or body fluid is provided. The method comprises the stepsof extracting and recovering the mycotoxin from the patient tissue orbody fluid, contacting the mycotoxin with an antibody directed againstthe mycotoxin, and identifying the myocotoxin. In another embodiment,the method can further comprise the step of quantifying the mycotoxin.In either of these embodiments, 1) the body fluid can be selected fromthe group consisting of urine, nasal secretions, nasal washes, bronchiallavages, bronchial washes, spinal fluid, sputum, gastric secretions,seminal fluid, other reproductive tract secretions, lymph fluid, wholeblood, serum, and plasma, 2) the mycotoxin can be selected from thegroup consisting of a gliotoxin and a patulin, 3) the tissue can bederived from a patient tissue biopsy and can be in a 10% formalinsolution or can be in a paraffin block, and/or 4) the antibody can bebound to a bead dyed with a fluorochrome. Any applicable combination of1 through 4 is also contemplated.

In yet another embodiment, a method of determining if a patient is atrisk for or has developed a disease state related to a yeast infectionis provided. The method comprises the steps of extracting and recoveringa yeast mycotoxin from a tissue or body fluid of the patient, contactingthe mycotoxin with an antibody directed against the toxin, identifyingthe mycotoxin, and determining if the patient is at risk for or hasdeveloped the disease state related to the yeast infection. In anotherembodiment, the method can further comprise the step of developing aneffective treatment regimen for the patient.

In either of the methods in the immediately preceding paragraph, 1) thebody fluid can be selected from the group consisting of urine, nasalsecretions, nasal washes, bronchial lavages, bronchial washes, spinalfluid, sputum, gastric secretions, seminal fluid, other reproductivetract secretions, lymph fluid, whole blood, serum, and plasma, 2) themycotoxin can be selected from the group consisting of a gliotoxin and apatulin, 3) the tissue can be derived from a patient tissue biopsy andcan be in a 10% formalin solution or can be in a paraffin block, and/or4) the antibody can be bound to a bead dyed with a fluorochrome. Anyapplicable combination of 1 through 4 is also contemplated.

In still another embodiment, a method of determining if a patient is atrisk for or has developed a disease state related to a yeast infectionis provided. The method comprises the steps of extracting and recoveringDNA of a specific yeast species from a tissue or body fluid of thepatient, amplifying the DNA, hybridizing a probe to the DNA tospecifically identify the yeast species, and specifically identifyingthe yeast species. In another embodiment, the method can furthercomprise the step of developing an effective treatment regimen for thepatient. In these two embodiments, the probe can be bound to a bead dyedwith a fluorochrome.

In another illustrative embodiment, a kit is provided comprising apurified nucleic acid with a sequence selected from the group consistingof SEQ ID NO: 1 to SEQ ID NO: 12 or with a complement of a sequenceselected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 12.

In yet another illustrative embodiment, a kit is provided comprisingcomponents for the extraction and recovery of a yeast mycotoxin frombody fluid or tissue of a patient. The kit can further comprisecomponents for identification of the mycotoxin. The components foridentification of the mycotoxin can include beads dyed with afluorochrome and coupled to antibodies to the mycotoxin or to themycotoxin or to a mycotoxin antigen.

In another embodiment, a purified nucleic acid is provided comprising asequence of SEQ ID NO: 1 to SEQ ID NO: 12 or a sequence that hybridizesunder highly stringent conditions to a sequence consisting of SEQ ID NO:1 to SEQ ID NO: 12.

In still another embodiment, a purified nucleic acid is providedcomprising a complement of a sequence of SEQ ID NO: 1 to SEQ ID NO: 12or a sequence that hybridizes under highly stringent conditions to acomplement of a sequence consisting of SEQ ID NO: 1 to SEQ ID NO: 12.

In another aspect, a method of detecting an antibody IP a mycotoxin in apatient body fluid is provided. The method comprises the steps ofcontacting the patient body fluid with a mycotoxin or a mycotoxinantigen coupled to a bead wherein the bead is dyed with a fluorochrome,and detecting the antibody.

In yet another aspect, a method of identifying a yeast species in apatient tissue or body fluid is provided. The method comprises the stepsof identifying a yeast mycotoxin in a patient tissue or body fluid, andspecifically identifying a yeast species in the mycotoxin positivepatient tissue or body fluid. The method can further comprise the stepsof extracting and recovering the mycotoxin from the patient tissue orbody fluid, and contacting the mycotoxin with an antibody directedagainst the mycotoxin. The method can further comprise the steps ofextracting and recovering DNA of the yeast species from the patienttissue or body fluid, amplifying the DNA, and hybridizing a probe to theDNA to specifically identify the yeast species.

In any of the embodiments in the immediately preceding paragraph, 1) theamplifying step can be performed with primers that hybridize to the DNA,2) the body fluids can be selected from the group consisting of urine,nasal secretions, nasal washes, bronchial lavages, bronchial washes,spinal fluid, sputum, gastric secretions, seminal fluid, otherreproductive tract secretions, lymph fluid, whole blood, serum, andplasma, 3) the DNA can be amplified using PCR, 4) the PCR can bereal-time PCR, 5) the probe can be fluorescently labeled, 6) the yeastspecies can be selected from the group consisting of Candida albicans,Candida glabrata, Candida kruseii, and Candida tropicalis, 7) theamplified sequence can be internal transcribed spacer regions of nuclearribosomal DNA, 8) the probe can be bound to a bead dyed with afluorochrome, 9) the method can further comprise the step of quantifyingthe mycotoxin, 10) the mycotoxin cm be selected from the groupconsisting of a gliotoxin and a patulin, 11) the tissue can be derivedfrom a patient tissue biopsy and can be in a 10% formalin solution or isin a paraffin block, and/or 12) the antibody can be bound to a bead dyedwith a fluorochrome. Any applicable combination of 1 through 12 is alsocontemplated.

DESCRIPTION OF THE ILLUSTRATIVE EMBODIMENTS

The present invention relates to methods and compositions foridentifying or detecting the presence of yeast in patient tissue andbody fluids. The identification and detection methods are based on 1.)amplification of yeast DNA using a PCR-based method; 2.) detection andquantification of mycotoxin in patient body fluids and tissues; and 3)combinations thereof. The methods and compositions (e.g., primers andprobes) for amplification of yeast DNA are highly specific and sensitiveand avoid co-amplification of or do not co-amplify non-specific human oranimal nucleic acids.

The methods and compositions for detection and quantification ofmycotoxins are also very specific and sensitive. These methods andcompositions utilize antibody-based identification of mycotoxins. Inillustrative embodiments, Enzyme Linked Immunosorbant Assay (ELISA), oraffinity chromatography can be used to detect mycotoxins produced byyeast. Illustratively, the mycotoxins can be gliotoxins or patulin.

In various illustrative embodiments, body fluids that can be tested forthe presence of yeast DNA or mycotoxins, or yeast DNA in combinationwith mycotoxins, include, but are not limited to, urine, nasalsecretions, nasal washes, inner ear fluids, bronchial lavages, bronchialwashes, alveolar lavages, spinal fluid, bone marrow aspirates, sputum,pleural fluids, synovial fluids, pericardial fluids, peritoneal fluids,saliva, tears, gastric secretions, stool, reproductive tract secretions,such as seminal fluid, lymph fluid, and whole blood, serum, or plasma.These samples can be prepared for testing as described herein. Invarious embodiments, tissue samples can include tissue biopsies ofhospital patients or out-patients and autopsy specimens. As used herein,the term “tissue” includes, but is not limited to, biopsies, autopsyspecimens, cell extracts, tissue sections, aspirates, tissue swabs, andfine needle aspirates.

In accordance with the invention the word “patient” means a human or ananimal, such as a domestic animal (e.g., a dog or a cat). Accordingly,the methods and compositions disclosed herein can be used for both humanclinical medicine and veterinary applications. Thus, the patientafflicted with a disease state related to a yeast infection can be ahuman, or in the case of veterinary applications, can be a laboratory,agricultural, domestic or wild animal. The present invention can beapplied to patients including, but not limited to, humans, laboratoryanimals such as rodents (e.g., mice, rats, hamsters, etc.), rabbits,monkeys, chimpanzees, domestic animals such as dogs, cats, and rabbits,agricultural animals such as cows, horses, pigs, sheep, goats, chickens,and wild animals in captivity such as bears, pandas, lions, tigers,leopards, elephants, zebras, giraffes, gorillas, dolphins, and whales.

The methods and compositions described herein can be used to detect oridentify microbial DNA (e.g., yeast DNA) or microbial toxins (e.g.,mycotoxins), or microbial DNA in combination with microbial toxins. Inembodiments where the microbe is a yeast species, the microbe istypically selected from the group consisting of various Candida species,for example, Candida albicans, Candida glabrata, Candida kruseii,Candida tropicalis, Candida stellatoidea, Candida parapsilosis, Candidaguilliermondii, Candida viswanathii, Candida pseudotropicalis, andCandida lusitaniae, as well as Rhodotorula mucilaginosa and Cryptococcusneoformans.

In one illustrative embodiment, a method is provided of identifying aspecific yeast species in a patient tissue or body fluid. The methodcomprises the steps of extracting and recovering DNA of the yeastspecies from the patient tissue or body fluid, amplifying the DNA,hybridizing a probe to the DNA to specifically identify the yeastspecies, and specifically identifying the yeast species.

In some embodiments, real-time PCR-based methods can be used to amplifythe yeast DNA and to detect and identify yeast DNA by hybridization ofthe probe to the yeast DNA. PCR is described in U.S. Pat. Nos. 4,683,202and 4,800,159, incorporated herein by reference, and methods for PCR arewell-known in the art. Real-time PCR combines amplification andsimultaneous probe hybridization to achieve sensitive and specificdetection of infectious yeast species in real-time thereby providinginstant detection of the yeast species. In this embodiment, the time todetect or identify the yeast and to obtain a diagnosis is greatlyreduced. Real-time PCR is conducted according to methods well-known inthe art. Exemplary probes and primers and their target DNAs, that can beused in accordance with the invention are shown below. “F1” refers to aforward primer, “R1” refers to a reverse primer, and “P1” refers to aprobe sequence which are well known terms in the art.

TABLE 1 Sequences and concentrations of primers and probes andcorresponding probe-specific T_(m) in real-time PCR assays 5′ SequenceDescription Mod Sequence 3′ Mod Purification Candida albicans CAP1 (SEQID NO: 1) 6FAM TCGGGGGCGGCCGCTGCGG BHQ #1 Dual HPLC CAF1 (SEQ ID NO: 2)AAAAAGTACGTGAAATTGTTG Stnd. Desalt CAR1 (SEQ ID NO: 3) AAGCCGTGCCACATTCStnd. Desalt Candida glabrata CGP1 (SEQ ID NO: 4) 6FAMACCTAGGGAATGTGGCTCTGCG BHQ #1 Dual HPLC CGF1 (SEQ ID NO: 5)TGGGCCAGCATCGGTTTTG Stnd. Desalt CGR1 (SEQ ID NO: 6)CCTAGATAACAAGTATCGCAG Stnd. Desalt Candida krusei CKP1 (SEQ ID NO: 7)6FAM AAGGCGGTGTCCAAGTCCCTTG BHQ #1 Dual HPLC CKF1 (SEQ ID NO: 8)TCAGTAGCGGCGAGTGAAG Stnd. Desalt CKR1 (SEQ ID NO: 9) AGAAGGGCCTCACTGCTTCStnd. Desalt Candida tropicalis CTP1 (SEQ ID NO: 10) 6FAMTCGGGGGTGGCCTCTACAG BHQ #1 Dual HPLC CTF1 (SEQ ID NO: 11)AAAAAGTACGTGAAATTGTTG Stnd. Desalt CTR1 (SEQ ID NO: 12) AAGCCGTGCCACATTCStnd. Desalt

In various embodiments, sample preparation (i.e., preparation of thetarget DNA) involves rupturing the cells (e.g., cells of the tissue oryeast in patient body fluid or tissue) and isolating the yeast DNA fromthe lysate. Techniques for rupturing cells and for isolation of DNA arewell-known in the art. For example, cells may be ruptured by using adetergent or a solvent, such as phenol-chloroform. DNA may be separatedfrom the lysate by physical methods including, but not limited to,centrifugation, pressure techniques, or by using a substance withaffinity for DNA, such as, for example, silica beads. After sufficientwashing, the isolated DNA may be suspended in either water or a buffer.In other embodiments, commercial kits are available, such as Quiagen™,Nuclisensm™, and Wizard™ (Promega), and Promegam™. Methods for isolatingDNA are described in Sambrook et al., “Molecular Cloning: A LaboratoryManual”, 3rd Edition, Cold Spring Harbor Laboratory Press, (2001),incorporated herein by reference.

In various embodiments described herein, the primers and probes used foramplification of the target DNA and for detection and identification ofyeast DNA are oligonucleotides from about ten to about one hundred, moretypically from about ten to about thirty or about six to abouttwenty-five base pairs long, but any suitable sequence length can beused. In illustrative embodiments, the primers and probes may bedouble-stranded or single-stranded, but the primers and probes aretypically single-stranded. The primers and probes described herein arecapable of specific hybridization, under appropriate hybridizationconditions (e.g., appropriate buffer, ionic strength, temperature,formamide, and MgCl₂ concentrations), to a region of the target DNA. Theprimers and probes described herein are designed based on having amelting temperature within a certain range, and substantialcomplementarity to the target DNA. Methods for the design of primers andprobes are described in Sambrook et al., “Molecular Cloning: ALaboratory Manual”, 3rd Edition, Cold Spring Harbor Laboratory Press,(2001), incorporated herein by reference.

The primers and probes described herein for use in PCR can be modifiedby substitution, deletion, truncation, and/or can be fused with othernucleic acid molecules wherein the resulting primers and probeshybridize specifically to the intended targets and are useful in themethods described herein for amplification of the target DNAs.Derivatives can also be made such as phosphorothioate, phosphotriester,phosphoramidate, and methylphosphonate derivatives, that specificallybind to single-stranded DNA or RNA (Goodchild, et al., Proc. Natl. Acad.Sci. 83:4143-4146 (1986)).

The invention encompasses isolated or substantially purified nucleicacids. An “isolated” or “purified” nucleic acid molecule issubstantially free of other cellular material, or culture medium whenproduced by recombinant techniques, or substantially free of chemicalprecursors or other chemicals when chemically synthesized. Preferably,an “isolated” or “purified” nucleic acid is free of sequences thatnaturally flank the nucleic acid in the genomic DNA of the organism fromwhich the nucleic acid is derived. For example, in various embodiments,the isolated or purified nucleic acid molecule can contain less thanabout 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotidesequences that naturally flank the nucleic acid molecule in genomic DNAof the cell from which the nucleic acid is derived.

Also within the scope of the invention are nucleic acids complementaryto the probes and primers described herein, and those that hybridize tothe nucleic acids described herein or those that hybridize to theircomplements under highly stringent conditions. In accordance with theinvention “highly stringent conditions” means hybridization at 65° C. in5×SSPE and 50% formamide, and washing at 65° C. in 0.5×SSPE. Conditionsfor low stringency and moderately stringent hybridization are describedin Sambrook et al., “Molecular Cloning: A Laboratory Manual”, 3rdEdition, Cold Spring Harbor Laboratory Press, (2001), incorporatedherein by reference. In some illustrative aspects, hybridization occursalong the full-length of the nucleic acid.

Also included are nucleic acid molecules having about 60%, about 70%,about 75%, about 80%, about 85%, about 90%, about 95%, 96%, 97%, and 98%homology to the probes and primers described herein. Determination ofpercent identity or similarity between sequences can be done, forexample, by using the GAP program (Genetics Computer Group, software;now available via Accelrys on http://www.accelrys.com), and alignmentscan be done using, for example, the ClustalW algorithm (VNTI software,InforMax Inc.). A sequence database can be searched using the nucleicacid sequence of interest. Algorithms for database searching aretypically based on the BLAST software (Altschul et al., 1990). In someembodiments, the percent identity can be determined along thefull-length of the nucleic acid.

As used herein, the term “complementary” refers to the ability of purineand pyrimidine nucleotide sequences to associate through hydrogenbonding to form double-stranded nucleic acid molecules. Guanine andcytosine, adenine and thymine, and adenine and uracil are complementaryand can associate through hydrogen bonding resulting in the formation ofdouble-stranded nucleic acid molecules when two nucleic acid moleculeshave “complementary” sequences. The complementary sequences can be DNAor RNA sequences. The complementary DNA or RNA sequences are referred toas a “complement.”

Techniques for synthesizing the probes and primers described herein arewell-known in the art and include chemical syntheses and recombinantmethods. Such techniques are described in Sambrook et al., “MolecularCloning: A Laboratory Manual”, 3rd Edition, Cold Spring HarborLaboratory Press, (2001), incorporated herein by reference. Primers andprobes can also be made commercially (e.g., CytoMol, Sunnyvale, Calif.or Integrated DNA Technologies, Skokie, Ill.). Techniques for purifyingor isolating the probes and primers described herein are well-known inthe art. Such techniques are described in Sambrook et al., “MolecularCloning: A Laboratory Manual”, 3rd Edition, Cold Spring HarborLaboratory Press, (2001), incorporated herein by reference. The primersand probes described herein can be analyzed by techniques known in theart, such as restriction enzyme analysis or sequencing, to determine ifthe sequence of the primers and probes is correct.

In various embodiments of the methods and compositions described herein,the probes and primers can be labeled, such as with fluorescentcompounds, radioactive isotopes, antigens, biotin-avidin, colorimetriccompounds, or other labeling agents known to those of skill in the art,to allow detection and quantification of amplified DNA, such as byReal-Time PCR. In illustrative embodiments, the labels may include6-carboxyfluorescein (FAM™), TET™ (tetrachloro-6-carboxyfluorescein),JOE™ (2,7,-dimethoxy-4,5-dichloro-6-carboxyfluorescein), VIC™, HEX(hexachloro-6-carboxyfluorescein), TAMRA™(6-carboxy-N,N,N′,N′-tetramethylrhodamine), BHQ™, SYBR® Green, Alexa350, Alexa 430, AMCA, BODIPY 630/650, BODIPY 650/665, BODIPY-FL,BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, Cascade Blue, Cy3, Cy5,6-FAM,Fluorescein, Oregon Green 488, Oregon Green 500, Oregon Green 514,Pacific Blue, REG, Rhodamine Green, Rhodamine Red, ROX, and/or TexasRed.

In one illustrative embodiment, universal probes can be used to providea method for determining the presence of yeast DNA before conductingtarget-specific assays. In one embodiment, universal probes and primerscan be used to detect the presence of Candida species.

In another illustrative embodiment, a method is provided of identifyinga mycotoxin in patient tissue or body fluid. The method comprises thesteps of extracting and recovering the mycotoxin from the patient tissueor body fluid, contacting the mycotoxin with an antibody directedagainst the mycotoxin, and identifying the myocotoxin.

In accordance with this invention, “mycotoxin” means a yeast mycotoxin,such as a gliotoxin or patulin.

Illustratively, patient (e.g., human or animal) tissue is received in1.) a 10% formalin fluid or 2.) in a paraffin block in which the tissuehas been fixed in formalin. In one embodiment for mycotoxin detectionand quantitation, the tissue can then be processed by variousdehydration steps and finally embedded in paraffin. In this embodiment,the tissue can then be cut in 3-5 micron samples. In an illustrativeembodiment, approximately 25-35 mg of tissue can then be processed asdescribed in Examples 2-6 for mycotoxin extraction. Illustratively, bodyfluids can be prepared as described in Examples 1 and 3-6 or by othermethods known in the art. In another illustrative embodiment, patientbody fluids can be tested for the presence of mycotoxins.Illustratively, any antigen associated with a yeast or with a mycotoxincan be detected.

In the embodiment where mycotoxins are identified and quantitated,control samples of the body fluid or tissue to be analyzed can beobtained from patients with no documented history of exposure to yeastor mycotoxins. For example, negative control samples can be obtainedfrom autopsy specimens in which the patient had no exposure tomycotoxins or yeast (e.g., victims of motor vehicle accidents, coronaryartery disease, or myocardial infarction). For positive controls, forexample, samples of negative tissue and/or body fluids can be spikedwith known positive amounts of mycotoxins or yeast prior to evaluationto generate a calibration curve.

In another embodiment, a method is provided of determining if a patientis at risk for or has developed a disease state related to a yeastinfection. The method comprises the steps of extracting and recovering amycotoxin (i.e., a mycotoxin or a mycotoxin antigen) from a tissue orbody fluid of the patient, contacting the mycotoxin (i.e., a mycotoxinor a mycotoxin antigen) with an antibody directed against the toxin,identifying the mycotoxin (i.e., a mycotoxin or a mycotoxin antigen) anddetermining if the patient is at risk for or has developed the diseasestate related to the yeast infection. In another embodiment, a method isprovided of determining if a patient is at risk for or has developed adisease state related to a yeast infection. The method comprises thesteps of extracting and recovering DNA of a specific yeast species froma tissue or body fluid of the patient, amplifying the DNA, hybridizing aprobe to the DNA to specifically identify the yeast species, andspecifically identifying the yeast species.

In any embodiment involving “determining if the patient has developedthe disease state related to the yeast infection,” this phrase means“diagnosing the patient with a yeast infection.”

The method embodiments described in the preceding paragraph providemethods of diagnosing yeast infections. Patients in need of diagnosis ofa yeast infection can include cancer patients, post-operative patients,transplant patients, patients undergoing chemotherapy, immunosuppressedpatients, and the like. Patients in need of diagnosis may include humansor animals.

In one embodiment, for diagnosing yeast infections, kits are provided.The kits are useful for identifying, detecting, or quantitating yeastDNA or mycotoxins, or yeast DNA in combination with mycotoxins, in apatient tissue or body fluid. In the embodiment where the kit is used toidentify yeast DNA, the kit can contain the probes and/or primersdescribed herein, components to extract and isolate yeast DNA, andcomponents for DNA amplification, such as a heat stable DNA polymerase(e.g., Taq polymerase or Vent polymerase), buffers, MgCl₂, H₂O, and thelike. In the embodiment where the kit is used to identify mycotoxins(i.e., a mycotoxin or a mycotoxin antigen), the kit can containcomponents to extract and isolate the mycotoxin (i.e., a mycotoxin or amycotoxin antigen), antibody affinity matrices, ELISA plates, Luminex®beads, polyclonal or monoclonal antibodies, color development reagents,buffers, and the like.

In one embodiment, the reagents can remain in liquid form. In anotherembodiment, the reagents can be lyophilized. In another illustrativeembodiment, the kit can be used to detect other yeast antigens. The kitscan also contain instructions for use.

In another embodiment, a kit is provided comprising a purified nucleicacid with a sequence as described in Table 1 or a complement of asequence as described in Table 1. The kit may include a nucleic acidselected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 12, orthe complement of any of these nucleic acids. “Selected from the groupconsisting of SEQ ID NO: 1 to SEQ ID NO: 12” means the group consistingof SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5,SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10,SEQ ID NO: 11, and SEQ ID NO: 12. The kit can comprise components forthe extraction and recovery of yeast DNA or a mycotoxin, or combinationsthereof, from the body fluid or tissue of a patient. The kit can furthercomprise components for identification of the yeast DNA or themycotoxin, or combinations thereof. The components for identification ofthe yeast DNA or the mycotoxin, or combinations thereof, can includebeads dyed with a fluorochrome and coupled to a probe for the yeast DNAor to antibodies to the mycotoxin or to the mycotoxin itself or to amycotoxin antigen.

A purified nucleic acid is also provided comprising a sequence asdescribed in Table 1 or a sequence that hybridizes under highlystringent conditions to a sequence as described in Table 1. Inaccordance with the invention “highly stringent conditions” meanshybridization at 65° C. in 5×SSPE and 50% formamide, and washing at 65°C. in 0.5×SSPE.

A calibration reagent (or multiple calibration reagents) can also beincluded in the mycotoxin kit and “calibration reagent” means anystandard or reference material containing a known amount of themycotoxin (i.e., a mycotoxin or a mycotoxin antigen). The samplesuspected of containing the mycotoxin and the calibration reagent (ormultiple calibration reagents) are assayed under similar conditions. Themycotoxin concentration is then calculated by comparing the resultsobtained for the unknown sample with the results obtained for thecalibration reagent(s).

In accordance with one embodiment of the invention, a method ofidentifying a yeast species in a patient tissue or body fluid isdescribed. A patient tissue or body fluid (e.g., blood, serum, spinalfluid, urine, sputum, or nasal washes) is first examined for thepresence of mycotoxins. The method comprises the steps of extracting andrecovering the mycotoxin from the patient tissue or body fluid,contacting the mycotoxin with an antibody directed against themycotoxin, and identifying the mycotoxin. If a mycotoxin is present, thespecimen is then tested for the presence of specific yeast species, themethod further comprising the steps of extracting and recovering DNA ofthe yeast species from the patient tissue or body fluid, amplifying theDNA, hybridizing a probe to the DNA to specifically identify the yeastspecies, and specifically identifying the yeast species.

In various illustrative embodiments, the identification of a mycotoxinin a patient tissue or body fluid is followed by steps to specificallyidentify a corresponding yeast species in the patient tissue or bodyfluid. For example, if Candida albicans, Candida glabrata, Candidakruseii, or Candida tropicalis are identified in the patient tissue orbody fluid, steps for specifically identifying, e.g., gliotoxins orother specific yeast mycotoxins will follow.

The following examples provide illustrative methods for carrying out thepractice of the present invention. As such, these examples are providedfor illustrative purposes only and are not intended to be limiting.

Example 1 Samples Clinical Samples—

Samples obtained for routine microbiology diagnostic procedures frompatients suspected and not suspected of having yeast infections wereobtained for PCR assays. Samples include sputum, urine, andEDTA-anticoagulated blood. Part of the material was cultured usingstandard cultivation methods and part was used for RT-PCR. All specimenswere stored at ±20° C. until used for DNA extraction.

Yeast Strains—

Yeast strains were obtained from the American Type Culture Collection(ATCC), Manassas, Va. The identification of all clinical isolates wasconfirmed by conventional morphological and physiological methods. DNAwas extracted from the following isolates: C. glabrata (ATCC 90030), C.tropicalis (ATCC 750), C. albicans (ATCC 44374), and C. krusei (ATCC6258).

Yeast isolates were cultured by standard cultivation methods. Cellsuspensions were prepared with 0.9% saline and adjusted to a 3 McFarlandstandard. The yeast suspensions were centrifuged, resuspended in 200 μl0.9% NaCl, and incubated with 20 U recombinant Lyticase (Sigma-Aldrich,)at 37° C. for 30 min.

Example 2 DNA Extraction from Blood

A modification of a protocol described by Loeffler et al. was used. Forred cell lysis, 3 ml EDTA-blood was mixed with 15 ml lysis buffer (LB;10 mM Tris [pH 7.6], 5 mM MgCl₂, 10 mM NaCl), incubated for 15 mM onice, and then centrifuged for 10 min at 3,000 rpm. The pellet wasresuspended in 15 ml LB, incubated again for 15 min on ice, and thencentrifuged for 10 mM at 3,000 rpm. For white cell lysis, the pellet wasthen resuspended in 1 ml LB containing 200 μg/ml protease (QIAGEN,Hilden, Germany), incubated at 65° C. for 45 min, and then centrifugedat 13,000 rpm for 10 min. To obtain spheroplasts, the pellet wasresuspended in 500 μl Lyticase solution (50 mM Tris [pH 7.6], 1 mM EDTA[pH 8.0], 0.2% 2-mercaptoethanol) containing 20 U recombinant Lyticase,incubated at 37° C. for 30 min, and then centrifuged at 13,000 rpm for10 min. Finally, DNA was extracted with a High Pure PCR templatepreparation kit by following the instructions of the manufacturer. DNAwas eluted with 100 μl elution buffer.

Example 3 DNA Extraction from Cerebrospinal Fluid (CSF) and Sputum

For concentration of the yeast, 1 to 2 ml of the specimen wascentrifuged for 5 min at 13,000 rpm. To obtain spheroplasts, the pelletplus 200 μl supernatant was incubated with 20 U recombinant Lyticase at37° C. for 30 min. Finally, DNA was extracted with a High Pure PCRtemplate preparation kit by following the instructions of themanufacturer. DNA was eluted with 100 μl elution buffer.

Example 4 DNA Extraction from Tissue

Tissue (0.02 g) was incubated in 200 μl elution buffer and 200 μlbinding buffer from the High Pure PCR template preparation kit with 900μg protease at 55° C. until the tissue was completely digested. Afterinactivation of the protease (95° C. for 5 min), the sample was treatedwith 20 U recombinant Lyticase at 37° C. for 30 min to obtainspheroplasts. Finally, DNA was extracted with a High Pure PCR templatepreparation kit by following the instructions of the manufacturer. DNAwas eluted with 100 μl elution buffer.

To avoid contamination in all extraction methods herein described, allsteps were performed with aerosol-resistant tips. DNA extraction,preparation of the master mix, and addition of the template were carriedout in two separate rooms. For each extraction, a reagent blank wascarried out to exclude false-positive PCR results due to contamination.

Example 5 Candida Extraction from Urine

Candida DNA was extracted from urine using a modified procedure fromNorgen's Urine Bacteria Genomic DNA Isolation Kit (Thorold, ON, Canada).The kit is designed for the rapid preparation of bacterial genomic DNAfrom 1-20 ml of urine. The modification here demonstrates that yeastgenomic DNA can be isolated from both human urine and animal urines inorder to study the levels and types of yeast that are present.

Purification is based on spin column chromatography (similar to that inQuiagen's procedure listed above to extract mold spore DNA). The processinvolved obtaining the urine and pelleting the yeast cells presentthrough centrifugation. The cells were then resuspended in aResuspension Buffer and vortexed. The cells were then lysed usinglysozyme, Proeinase K and a Lysis Solution. The lysate was applied tothe spin columns containing resin. The resin binds DNA in a manner thatdepends on ionic concentrations. Thus, only the DNA will bind to thecolumn while most of the RNA and digested proteins are removed. Thebound DNA was washed twice with the two provided Wash Solutions in orderto remove any impurities. Finally, the genomic DNA was eluted with theElution Buffer. The purified DNA is of the highest quality and can beused in a number of applications. The time to complete the purificationprocess is approximately 45 minutes.

Procedure:

-   1. Transfer 1.5 ml of urine to a microcentrifuge tube and centrifuge    at 13,000×g for 4 minutes to pellet the cells. Pour off supernantant    so as not to disturb the cell pellet.-   2. Add 250 ul of Resupension Solution (Norgen) to cell pellet and    vortex gently.-   3. Add 12 ul of lysozyme stock solution.-   4. Add 250 ul of Lysis Solution and 12 ul of Proeinase K to the cell    suspension. Mix by gentle vortexing and incubate at 55 degrees C.    for 30 minutes.-   5. Add 60 ul of Binding Solution to the lysate and mix by gently    vortexing.-   6. Assemble a micro spin column with a provided collection tube.    Apply the mixture to the spin column assembly.-   7. Centrifuge the unit for 3 minutes at 6000×g (8000 RPM).-   8. Apply 500 ul of Wash Solution I to column, centrifuge the unit    for 3 minutes at 13000×g.-   9. After centrifugation, discard the flowthrough and reassemble the    spin column with its collection tube.-   10. Apply 500 ul of Wash Solution II to the column.-   11. Centrifuge for 3 minutes at 13000×g.-   12. Detach the spin column from the collection tube and discard the    collection tube and flow through.-   13. Assemble the spin column (with DNA bound to the resin) with a    1.7 ml Elution tube.-   14. Add 100 ul of Elution Buffer to the center of the resin bed.-   15. Centrifuge for 1 minute at 3000 g. A portion of the Elution    Buffer will pass through the column which allows for hydration of    the DNA to occur.-   16. Centrifuge at 13000×g for an additional 2 minutes to collect the    total elution volume-   17. The purified DNA can be stored at 2-8 degrees C. for 3 days or    stored at −20 degrees C. for an extended amount of time.

Example 6 Validation of Candida DNA

Novel real-time PCR assays targeting various yeast species, includingCandida species (for example, Candida albicans, Candida glabrata,Candida krusei, and Candida tropicalis) are herein described. The assayscan be performed either as single assays or simultaneously using aCepheid Smart Cycler. Specimens were evaluated using pure cultures andEDTA-anticoagulated blood, cerebrospinal fluid (CSF), and urine spikedwith C. albicans, C. glabrata, C. krusei, anti C. tropicalis cellsuspensions. Yeast Genomic DNA was isolated using NORGEN Urine BacteriaGenomic DNA Isolation Kit, Ontario Canada.

The validation showed that the Cepheid SmartCycler System utilizingspecially designed target probes and primers can detect yeast inclinical fluid and urine utilizing real-time PCR technology. Thevalidation showed that the SmartCycler can generate a qualitativedetermination of the presence or absence of yeast species in clinicalsamples for various targets, for example:

Candida C. albicans C. glabrata C. kruseii C. tropicalis

Example 7 Description of Smart Cycler System

The RT-PCR assays as used herein employ a closed system using theCepheid Smart Cycler which decreases the risk of false-positive results.Fast turnaround time is also significant in using RT-PCR. The SmartCycler system is a real-time PCR instrument with rapid cycling times andrandom access capabilities. Four-color detection allows for multiplexreactions. Cepheid's single test, disposable reaction tubes were used.As testing volumes expand, the SmartCycler system can be configured foradditional testing with 16, 32, 48, 64, 80, and 96 reaction sitesystems. The integrated software system allows for the performance ofCepheid kits or user developed assays.

The system is fully programmable and can be adapted to run theprobe/primer set utilized in the targets being validated. The followingrepresents the general flow of a clinical sample from extraction toreporting.

The clinical sample is extracted utilizing a commercial extraction kit.After the extraction, PCR reactions are setup with the extracted nucleicacid from the clinical sample (and positive and negative control) andeach of the target probe and primer set. The reactions are placed in theSmartCycler and a preprogrammed real-time program is run for eachreaction with data being collected by the instrument. After theinstrument runs are complete, the data from each run is analyzed todetermine if the run is acceptable and to determine if the clinicalsample is positive or negative for the target tested. Upon completion ofthis analysis, a report is generated for the sample processed and theresults of the testing is reviewed, approved, and reported.

A run is determined to be acceptable if the internal control for theextracted sample is positive, the positive target control is positive,the negative target control is negative, and no instrument errors aregenerated during the course of a PCR run.

Example 8 Validation Protocol Test Samples Required:

Yeast Target Samples: show that all samples can be detected utilizingtarget specific probes.

Yeast Target Samples: Stock suspensions of each target and serialdilutions based on the estimated concentration of the stock suspension.

Target 1st Point 2nd Point 3rd Point Stock 1:10 Dil 1:10 Dil

Clinical Urine Samples: Negative urine samples spiked with the targetand spores for the internal control Geometrica.

Negative Patient Samples: Patient urine samples expected to be negativefor yeast (Used for negative controls).

Testing Conditions:

Real-time PCR was performed on the extracted target suspension stocksutilizing the Candida Assays to provide a cross over point for the stocksolutions.

Real-time PCR was performed on the extracted target suspensions and theGeometrica internal control spores (dilutions shown in the chart above)with the appropriate probe/primer. PCR will also be performed on anegative tissue control for the target. The dilutions will be processedin triplicate with the target primer/probe set.

Real-time PCR was performed on the extracted target-spiked urine samplesutilizing the target probe/primer set and the internal controlprobe/primer set. PCR will also be performed on a positive control forthe target and a negative urine control for the target.

Real-time PCR was performed to show specificity from assay to assayutilizing all Candida assays run with all Candida stocks.

Data Collected:

The Real-time PCR results of the target suspension stocks.

The real-time PCR results of the extracted target suspension dilutions(run in triplicate) with the appropriate probe/primer. PCR results of anegative urine control (run in triplicate).

The real-time PCR results of the extracted target-spiked urine utilizingthe target probe/primer set and the internal control probe/primer set.Also, results from the positive control for the target and a negativeurine control for the target.

The real-time PCR results showing specificity from assay to assayutilizing all Candida assays with all Candida stocks.

Example 9 Acceptance Criteria Definitions:

Positive Result: A positive result is defined as any amplificationobserved crossing a baseline fluorescence of ≧30 between cycles 1 and 45of the real-time PCR run.

Negative Result: A negative result is defined as no amplificationobserved crossing a baseline fluorescence of ≧30 between cycles 1 and 45of the PCR run.

Equivocal Result: An equivocal result is defined as no amplificationobserved crossing a baseline fluorescence of ≧30 between cycles 1 and45, a control out of range or questions regarding sample integrity.

Positive Control: A control that is positive for the target being testedand shows that the assay will show a positive in the presence of targetDNA and that there is not PCR inhibition. (Note: a sample that showsamplification for a target when the positive control is negative can bereported as a positive result.)

Negative Control: A control that is negative for the target being testedand shows that the reagents or the sample were not contaminated with thetarget prior to the testing of the sample. (Note: a sample that showsamplification at an earlier cycle than a contaminated negative controlcan be reported as a positive due to the fact that the contaminationcannot cause a sample to report a stronger positive than thecontamination.)

Internal Control: A control used to show that the extraction process isworking fine for the purification of nucleic acid from the clinicalspecimen and that a negative result is truly negative and not due to anissue associated with the extraction. (Note: the internal control mustbe positive for any sample to be reported as negative for a target.)

Target suspension stocks: Target stocks tested produces a positiveresult when run with the appropriate probe/primer set.

Target suspension Dilution and Precision: Target stocks tested producesa positive result when run with the appropriate probe/primer set foreach dilution point (in triplicate) in a real-time PCR run. Resultsdemonstrate the ability to detect each target at differingconcentrations with precision in each of three reactions.

Extracted target-spiked urine samples: The spiked clinical urine sampletested produces a positive result when tested with the targetprobe/primer set (including the Geometrica internal control set) in areal-time PCR run. The negative control is negative, the positivecontrol is positive, and the internal control is positive as expected.

Target Specificity: Results show that each assay is specific to thetarget that the assay is used to detect. This was demonstrated byrunning each assay with all target stocks with only the appropriatestocks giving a positive result when run with the appropriate assay.

Example 10 Validation Results

TABLE 2 Target Stocks Assay MSCI Run Target # Sample ID Result ResultNumber C kruseii 1 kruseii Stock Positive OK 101007.1 C tropicalis 2tropicalis Stock Positive OK 101007.1 C glabrata 3 glabrata StockPositive OK 101007.1 C albicans 4 albicans Stock Positive OK 101007.1

TABLE 3 Target Detection and Precision Stock 1:10 Dil 1:10 Dil AssayLoad (1^(st) (2^(nd) (3^(rd) Run Target # # point) Point) Point) NumberC krusei 1 1 15.47 19.40 22.50 102207.3 2 15.80 19.24 22.58 102207.3 316.18 19.35 na 102207.3 C tropicalis 2 1 16.62 19.54 23.07 102207.3 216.25 19.74 22.94 102207.3 3 16.32 19.72 na 102207.3 C glabrata 3 113.78 17.51 20.76 102207.4 2 14.20 17.74 21.07 102207.4 3 14.24 17.75 Na102207.4 C albicans 4 1 14.20 17.30 20.72 102207.4 2 14.10 17.32 20.54102207.4 3 14.16 17.44 na 102207.4

TABLE 4 Urine Samples Unknown Organism expected in Positive NegativeOrganism Sample Urine Samples Assay Run Control Control Geo Detected P3C albicans C glabrata Not Run Not Run Not Run Not Run Not Run P3 Calbicans C albicans Positive Negative Positive Positive 102307.3 P3 Calbicans C kruseii Not Run Not Run Not Run Not Run Not Run P3 C albicansC tropicalis Positive Negative Positive Not Detected 102307.2 P4 Ctropicalis C glabrata Not Run Not Run Not Run Not Run Not Run P4 Ctropicalis C albicans Not Run Not Run Not Run Not Run Not Run P4 Ctropicalis C kruseii Not Run Not Run Not Run Not Run Not Run P4 Ctropicalis C tropicalis Positive Negative Positive Positive 102307.2 P5C kruseii C glabrata Not Run Not Run Not Run Not Run Not Run P5 Ckruseii C albicans Not Run Not Run Not Run Not Run Not Run P5 C kruseiiC kruseii Positive Negative Positive Positive 102307.2 P5 C kruseii Ctropicalis Positive Negative Positive Not Detected 102307.2 P6 Cglabrata C glabrata Positive Negative Positive Positive 102307.3 P6 Cglabrata C albicans Not Run Not Run Not Run Not Run Not Run P6 Cglabrata C kruseii Not Run Not Run Not Run Not Run Not Run P6 C glabrataC tropicalis Positive Negative Positive Not Detected 102307.2 P9 Calbicans C glabrata Not Run Not Run Not Run Not Run Not Run P9 Calbicans C albicans Positive Negative Positive Positive 102307.3 P9 Calbicans C kruseii Not Run Not Run Not Run Not Run Not Run P9 C albicansC tropicalis Not Run Not Run Not Run Not Run Not Run P10 C kruseii & Cglabrata C glabrata Positive Negative Positive Positive 102307.3 P10 Ckruseii & C glabrata C albicans Not Run Not Run Not Run Not Run Not RunP10 C kruseii & C glabrata C kruseii Positive Negative Positive Positive102307.2 P10 C kruseii & C glabrata C tropicalis Not Run Not Run Not RunNot Run Not Run P11 C albicans & C glabrata C glabrata Positive NegativePositive Not detected 102307.3 P11 C albicans & C glabrata C albicansPositive Negative Positive Positive 102307.3 P11 C albicans & C glabrataC kruseii Not Run Not Run Not Run Not Run Not Run P11 C albicans & Cglabrata C tropicalis Not Run Not Run Not Run Not Run Not Run P12 Ctropicalis (CAP PT) C glabrata Not Run Not Run Not Run Not Run Not RunP12 C tropicalis (CAP PT) C albicans Not Run Not Run Not Run Not Run NotRun P12 C tropicalis (CAP PT) C kruseii Not Run Not Run Not Run Not RunNot Run P12 C tropicalis (CAP PT) C tropicalis Positive NegativePositive Positive 102307.2 P13 Negative C glabrata Positive NegativePositive Not Detected* 102307.3 P13 Negative C albicans PositiveNegative Positive Not Detected* 102307.3 P13 Negative C kruseii PositiveNegative Positive Not Detected 102307.2 P13 Negative C tropicalisPositive Negative Positive Not Detected* 102307.2

TABLE 5 Specificity C kruseii C albicans C tropicalis C glabrata RunSample Tested Assay Assay Assay Assay Number C kruseii (516) PositiveNot Detected Not Detected Positive* 021308.1 C tropicalis (517) NotDetected Not Detected Positive Positive* 021308.1 C glabrata (518) NotDetected Not Detected Not Detected Positive 021308.1 C albicans (519)Not Detected Positive Not Detected Positive* 021308.1

Four Candida Real-time PCR assays were run on extracted targetsuspension stocks. Each assay for each target was run on targetsolutions containing a specific organism and all assays producedpositive results.

Four Candida Real-time PCR assays were run on extracted targetsuspension stocks. Each assay provided positive results for eachdilution in triplicate demonstrating the ability to detect a target bydiffering concentrations in a precise and repetitive manner.

Four Candida Real-time PCR assays were run on spiked clinical urinesamples. All assays were able to detect the expected targets with theexception of sample P11. Sample P11 was supposed to contain both C.albicans and C. glabrata but the assays were only able to detect C.albicans. Based on the overall data generated for the C. glabrata assayincluding the spiked sample number P6 and P10 it is deduced that therewas either no or not adequate C. glabrata target present in the spikedsample number P11 to allow detection. The negative urine was negative asexpected.

Four Candida Real-time PCR assays were tested for specificity with allassays showing great specificity with the exception of C. glabrata. C.glabrata showed some level of detection on all sample targets with thestrongest detection for C. glabrata as expected. The weak detection ofC. glabrata on the other three sample stocks is most likely due to someC. glabrata contamination in the negative urine used for the spikingexperiment.

All four Candida assays passed the validation in exact accordance withthe acceptance criteria. These four target assays can be consideredvalidated for the qualitative determination of the presence or absenceof Candida in clinical urine samples.

Methods and compositions, such as those for PCR, as described in WO2008/051285, incorporated herein by reference, can also be used.

Example 11 Detection of Yeast in Clinical Samples Utilizing Real-TimePCR Technology

PCR analysis was performed as described above using clinical samples.Specifically, urine samples from 10 patients were analyzed using PCRassays designed to target various yeast species, including Candidaalbicans, Candida tropicalis, Candida glabrata, and Candida kruseii.

The target probes and primers used to detect various yeast species inthe urine samples utilizing real-time PCR technology were as follows:

Candida C. albicans C. tropicalis C. glabrata C. kruseii

Patients tested included women with asymptomatic urinary tractinfections (i.e., patients with a history of recurring yeast urinarytract infections, with no present urinary complaints). All patientstested had a history of recurring yeast infections with the most commonorganism being Candida albicans (by culture history).

PCR Results:

70% of patients tested (who, at present, had no history of urinaryinfection) had Candida albicans and Candida glabrata present in theurine.

30% of patients tested (who, at present, had no history of urinaryinfection) had no findings of yeast in the urine with the four probestested.

Typically, Candida glabrata is multiply resistant to many antifungalagents which are commonly used in symptomatic, culture positive urinaryinfections. Thus, the identification of this organism in a patientsample, will allow more powerful antifungal agents to be administered toalleviate the C. glabrata infection in the patient. In addition, thepowerful antifungal will alleviate the other less resistant yeastspecies present in the urine.

1. A method of identifying a specific yeast species in patient tissue orbody fluid, the method comprising the steps of: extracting andrecovering DNA of the yeast species from the patient tissue or bodyfluid; amplifying the DNA; hybridizing a probe to the DNA tospecifically identify the yeast species; and specifically identifyingthe yeast species.
 2. The method of claim 1 wherein the amplifying stepis performed with primers that hybridize to the DNA.
 3. The method ofclaim 1 wherein the body fluid are selected from the group consisting ofurine, nasal secretions, nasal washes, bronchial lavages, bronchialwashes, spinal fluid, sputum, gastric secretions, seminal fluid, otherreproductive tract secretions, lymph fluid, whole blood, serum, andplasma.
 4. The method of claim 1 wherein the DNA is amplified using PCR.5. The method of claim 4 wherein the PCR is real-time PCR.
 6. The methodof claim 1 wherein the probe is fluorescently labeled.
 7. The method ofclaim 5 wherein the probe is fluorescently labeled.
 8. The method ofclaim 1 wherein the yeast species are selected from the group consistingof Candida albicans, Candida glabrata, Candida kruseii, and Candidatropicalis.
 9. The method of claim 2 wherein the probe, a forwardprimer, and a reverse primer are used during the amplification step andthe probe comprises the sequence of SEQ ID NO: 1, the forward primercomprises the sequence of SEQ ID NO: 2, and the reverse primer comprisesthe sequence of SEQ ID NO:
 3. 10. The method of claim 2 wherein theprobe, a forward primer, and a reverse primer are used during theamplification step and the probe comprises the sequence of SEQ ID NO: 4,the forward primer comprises the sequence of SEQ ID NO: 5, and thereverse primer comprises the sequence of SEQ ID NO:
 6. 11. The method ofclaim 2 wherein the probe, a forward primer, and a reverse primer areused during the amplification step and the probe comprises the sequenceof SEQ ID NO: 7, the forward primer comprises the sequence of SEQ ID NO:8, and the reverse primer comprises the sequence of SEQ ID NO:
 9. 12.The method of claim 2 wherein the probe, a forward primer, and a reverseprimer are used during the amplification step and the probe comprisesthe sequence of SEQ ID NO: 10, the forward primer comprises the sequenceof SEQ ID NO: 11, and the reverse primer comprises the sequence of SEQID NO:
 12. 13. The method of claim 2 wherein the amplified sequence isinternal transcribed spacer regions of nuclear ribosomal DNA.
 14. Themethod of claim 1 wherein the probe is bound to a bead dyed with afluorochrome.
 15. A method of identifying a yeast mycotoxin in patienttissue or body fluid, the method comprising the steps of: extracting andrecovering the mycotoxin from the patient tissue or body fluid;contacting the mycotoxin with an antibody directed against themycotoxin; and identifying the myocotoxin.
 16. The method of claim 15further comprising the step of quantifying the mycotoxin.
 17. The methodof claim 15 wherein the body fluid is selected from the group consistingof urine, nasal secretions, nasal washes, bronchial lavages, bronchialwashes, spinal fluid, sputum, gastric secretions, seminal fluid, otherreproductive tract secretions, lymph fluid, whole blood, serum, andplasma.
 18. The method of claim 15 wherein the mycotoxin is selectedfrom the group consisting of a gliotoxin and a patulin.
 19. The methodof claim 15 wherein the tissue is derived from a patient tissue biopsyand is in a 10% formalin solution or is in a paraffin block.
 20. Themethod of claim 15 wherein the antibody is bound to a bead dyed with afluorochrome.
 21. A kit comprising a purified nucleic acid with asequence selected from the group consisting of SEQ ID NO: 1 to SEQ IDNO: 12 or with a complement of a sequence selected from the groupconsisting of SEQ ID NO: 1 to SEQ ID NO:
 12. 22. A kit comprisingcomponents for the extraction and recovery of a yeast mycotoxin frombody fluid or tissue of a patient.
 23. The kit of claim 22 furthercomprising components for identification of the mycotoxin.
 24. The kitof claim 23 wherein the components for identification of the mycotoxininclude beads dyed with a fluorochrome and coupled to antibodies to themycotoxin or to the mycotoxin or to a mycotoxin antigen.
 25. A purifiednucleic acid comprising a sequence of SEQ ID NO: 1 to SEQ ID NO: 12 or asequence that hybridizes under highly stringent conditions to a sequenceconsisting of SEQ ID NO: 1 to SEQ ID NO: 12.